We have focused our efforts to elucidate: i) the catalytic cycle and transport pathway of human Pgp; ii) the molecular basis of the polyspecificity of Pgp; iii) the interaction of clinically relevant tyrosine kinase inhibitors (TKIs) with Pgp and ABCG2; iv) determination of the binding site of nilotinib, a second generation TKI, on Pgp; v) pharmacophore features required for binding of nilotinib to Pgp and ABCG2; and vi) the fate of cell surface Pgp, its folding and stability. We have been employing cell-based, biochemical, biophysical, pharmacological, and physiological techniques along with molecular biology and molecular modeling approaches to extend our understanding of the mechanistic aspects and the structure-function relationships of ABC drug transporters. In addition, we have devoted considerable efforts to the screening and development of TKIs and small molecule modulators for Pgp and ABCG2. We found that several tyrosine kinase inhibitors, which are used in the clinic for treatment of various types of cancers, are either transport substrates or inhibitors of Pgp and/or ABCG2. 1. Elucidation of the catalytic cycle of ATP hydrolysis and transport pathway of Pgp and the role of conserved motifs in the ATP-binding cassette: We are continuing our studies on the catalytic cycle and transport pathway of Pgp. We are using molecular modeling and mutagenesis approaches to elucidate on a molecular level how this transporter recognizes and transports a wide variety of structurally dissimilar compounds. We have begun to use tmFRET, which is a novel biophysical method developed to determine short range (5 - 25 angstrom) distances within different locations of the protein at very low concentrations. Using this sensitive fluorescence-based method, we have begun to determine the changes in distance associated with the apo and the closed (ATP/Vi trapped) conformations of Pgp. With tmFRET, preliminary results show that there is a significant change in the distance of the two NBDs between the apo and closed conformations ( 20 angstrom). Similarly, results of disulfide crosslinking studies with the oxidant copper phenanthroline and bi-functional sulfhydryl group reagents indicate that human Pgp is a very flexible molecule and its NBDs are much closer to each other in the apo form. The distance between the C431 and C1074 residues in the Walker A motif of NBDs ranges from 5 to 25 angstrom in the apo (in the absence of ATP and drug-substrate) conformation. 2. Mechanism of the drug-mediated inhibition of Pgp ATPase activity. Most of the substrates or modulators of Pgp stimulate its basal ATPase activity, and only a few drugs have been found to inhibit it. Zosuquidar, tariquidar and elacridar, high affinity inhibitors of transport function, also inhibit Pgp ATPase activity, while a variety of substrates including verapamil, paclitaxel and vinblastine stimulate ATP hydrolysis. The molecular mechanisms that are in play, in either case (stimulation or inhibition), remain elusive. The development of an effective Pgp inhibitor certainly would benefit from the understanding of drug-mediated inhibition of ATP hydrolysis. Using directed mutagenesis we identified a pair of phenylalanine-tyrosine structural motifs of Pgp that are critical for the inhibition of ATP hydrolysis by high-affinity modulators. These structural motifs are located at the drug-binding pocket of Pgp. We found that drugs that inhibit the ATPase activity switch to stimulating the ATPase activity when any of these residues are mutated. For instance, zosuquidar inhibits the basal ATP hydrolysis of cysless WT Pgp with high affinity (IC50 = 10 nM). The inhibition is completely lost upon mutation of Y953 to alanine and is switched to stimulation when three polar residues are mutated (Y307A/Q725A/Y953A). Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with the tyrosines Y953 and Y310, respectively, in an edge-to-face conformation, helping thetyrosines to adopt the proper orientation to effectively establish hydrogen-bond contact with the inhibitors. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport. Upon mutation, they bind at lower affinity sites that lead to stimulation of ATP hydrolysis and a poor inhibition of transport. 3. Resolution of the three-dimensional structure of human Pgp: The resolution of the three-dimensional structure of Pgp is an ongoing project and for this we have developed a purification scheme that has yielded total protein of 7.5-10.0 mg of 99% homogeneously pure Pgp. Due to the flexible nature of human Pgp and the difficulty of generating crystals of good diffraction quality, we are also using single particle analysis by the cryo-electron microscopy technique. The current studies indicate that the structural features of human Pgp in the presence and absence of a Fab of conformation-sensitive monoclonal antibody can be observed at 15- to 20-angstrom resolution. We are optimizing conditions to reach a sub-nanometer resolution to obtain the structure of human Pgp in at least three different (apo, ADP-vanadate trapped and Fab-bound) conformations. The structural studies are carried out in collaboration with Drs. Di Xia and Sriram Subramanian. In addition, we have reconstituted human Pgp in giant unilamellar liposomes. Giant unilamellar liposomes (vesicles) are very useful to study the biophysical and kinetic properties of transport by Pgp, as these liposomes can be observed individually by optical microscopy. In collaboration with Dr. Michael Mayer (Univ. of Michigan), we reconstituted purified human Pgp into giant liposomes using a hydrogel method. Pgp in the giant liposomes was reconstituted with inside-out and right side out (same as in intact cells) orientations at equal levels, and it exhibited substrate-stimulated ATPase activity and ATP -dependent rhodamine 123 uptake. 4. Role of intracellular loops 1 and 3 in folding and stability of human Pgp: We investigated the role of residues in intracellular loops 1 and 3 in folding and maturation of human Pgp. To gain insight into the stability of cell surface Pgp, we assessed the degradation of cell surface Pgp. We characterized the pathway involved in degradation of Pgp following its internalization. We found that the half-life of Pgp at the cell surface in the colon cancer cell line HCT-15 is in the range of 26-27 h and treatment with the lysosomal inhibitor bafilomycin results in prolonged retention at the cell surface (half-life 35-36 h), indicating that the cell surface Pgp is degraded in lysosomes. Consistent with these results, internalized Pgp was found localized to the lysosomes. When cells were treated with the proteasomal inhibitors MG132 or lactacystin, the half-life of the protein was not altered, suggesting that the proteasomal pathway does not play a significant role in the degradation of cell surface Pgp. These studies may provide one or more therapeutic targets for the reversal of drug resistance by accelerating the degradation of cell surface transporters. To further explore the role of ICL 3 residues in folding, we mutated conserved R798, D800, D805, D806 and K808 to alanine and the mutant Pgps were expressed in HeLa cells using bac-mam baculovirus. We found that the cell surface expression of R798A, D800A, D805A and D806A was greatly reduced, and these mutants were retained in the ER. As expected, the mutant proteins were rescued to the cell surface by treatment with cyclosporine A and they were fully functional. However, the expression of K808A was not affected, and the mutation had no effect on transport function. Thus, the interaction of ICL3 and NBD2 is critical for folding of Pgp, but not for its function.